The CMS19 disease model specifies a pivotal role for collagen XIII in bone homeostasis

Mutations in the COL13A1 gene result in congenital myasthenic syndrome type 19 (CMS19), a disease of neuromuscular synapses and including various skeletal manifestations, particularly facial dysmorphisms. The phenotypic consequences in Col13a1 null mice (Col13a1−/−) recapitulate the muscle findings of the CMS19 patients. Collagen XIII (ColXIII) is exists as two forms, a transmembrane protein and a soluble molecule. While the Col13a1−/− mice have poorly formed neuromuscular junctions, the prevention of shedding of the ColXIII ectodomain in the Col13a1tm/tm mice results in acetylcholine receptor clusters of increased size and complexity. In view of the bone abnormalities in CMS19, we here studied the tubular and calvarial bone morphology of the Col13a1−/− mice. We discovered several craniofacial malformations, albeit less pronounced ones than in the human disease, and a reduction of cortical bone mass in aged mice. In the Col13a1tm/tm mice, where ColXIII is synthesized but the ectodomain shedding is prevented due to a mutation in a protease recognition sequence, the cortical bone mass decreased as well with age and the cephalometric analyses revealed significant craniofacial abnormalities but no clear phenotypical pattern. To conclude, our data indicates an intrinsic role for ColXIII, particularly the soluble form, in the upkeep of bone with aging and suggests the possibility of previously undiscovered bone pathologies in patients with CMS19.


Results
Diaphyseal cortical bone area and thickness are reduced in aged Col13a1 −/− and Col13a1 tm/tm mice. To determine the effects of absence of ColXIII in tubular bone morphometry, femurs of male and female Col13a1 −/− and Col13a1 tm/tm mice were imaged using µCT. Some reduction of bone mass could be depicted in the 72-week-old female Col13a1 −/− and Col13a1 tm/tm samples compared to their respective WT control samples in single mid-diaphyseal axial images ( Figs. 1 and 2). The 2D morphometric analysis showed a significant reduction of the cortical bone and the medullar area in the female Col13a1 −/− mice at the 72-week time point (Fig. 3A,B). In the Col13a1 tm/tm mice, the cortical bone area and cortical thickness were reduced in the 35-weekold as well as 72-week-old female mice (Fig. 4A,C). Additionally, the cortical bone and the medullar areas were reduced in the 72-week-old male Col13a1 tm/tm mice compared to the respective WT controls (Fig. 4A,B).
3D-morphometric analysis of distal femoral trabecular bone did not show major alterations in trabecular architecture or volume in the Col13a1 −/− or the Col13a1 tm/tm mice compared to the respective WT control samples (Figs. 3D-G, 4D-G). The differences in the trabecular architecture between sexes and phenotypes were quite evident in the 3D reconstruction images of distal femoral trabecular bone and are consistent with the 3D analysis Cortical bone area and thickness were reduced in the metaphyseal region in Col13a1 −/− and Col13a1 tm/tm mice. To confirm our findings in the Col13a1 −/− and the Col13a1 tm/tm mice, a high-resolution µCT scanner was utilized to re-analyse the 35-and 72-week-old female Col13a1 −/− and Col13a1 tm/tm mouse femur samples (Fig. 5). Scanner limitations enabled imaging only the distal third of the diaphysis, the metaphysis, and the distal growth plate to fit into a single image acquisition sequence. Thus, cortical bone was analyzed from the metaphyseal region. The cortical bone area and thickness were comparable to controls at the 35-week time point but significantly decreased in the 72-week-old Col13a1 −/− female mice (Fig. 6A,B) and in the 35-and 72-week-old Col13a1 tm/tm mice (Fig. 6D,E) while the medullary area was unaltered in both genotypes (Fig. 6C,F).
In the 72-week-old female Col13a1 −/− mice, the distal metaphyseal cortical bone area was 16% less and the cortical thickness 19% less compared to WT (Fig. 6A,B). In the female Col13a1 tm/tm mice, the distal metaphyseal cortical bone area was 9% less and the cortical thickness 8% less at the 35-week time point and 14% and 10% less, respectively, at the 72-week time point compared to WT controls (Fig. 6D,E). www.nature.com/scientificreports/ ; the whiskers represent min to max; *q < 0.05, **q < 0.01, ***q < 0.001 determined by two-way ANOVA and followed by the false discovery rate. www.nature.com/scientificreports/ ; the whiskers represent min to max; *q < 0.05, **q < 0.01, ***q < 0.001 determined by two-way ANOVA and followed by the false discovery rate. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Single axial and sagittal 2D projection images were generated from the 3D-reconstructed image datasets. From these projection images (Fig. 7), the morphometric analysis (Supplementary Table S1   www.nature.com/scientificreports/ were increased in the male Col13a1 tm/tm mice compared to WT control mice. No significant differences were found in the female Col13a1 tm/tm mice at the 4-or 35-week time points compared to the respective WT controls (Supplementary Table S3).

Discussion
In this study, we show that the femoral cortical bone area and cortical thickness of Col13a1 −/− and Col13a1 tm/tm mice are reduced in respect to control mice. ColXIII is a membrane-associated collagen existing as transmembrane and shed forms. Previous studies suggest that these two forms have distinct physiological functions 23,25,26 .
The role of ColXIII in bone homeostasis has been studied in a transgenic overexpression mouse model (Col1 3a1 oe ) 8,12,27 . In the Col13a1 oe mice, excess ColXIII causes a gradual increase of long and calvarial bone mass 27 , disrupts osteoblast differentiation, increases the expression of VEGF in primary osteoblasts and stimulates vascular outgrowth in cultured fetal mouse metatarsals 12 . The pronounced diaphyseal appositional bone mass phenotype is accompanied by endocortical bone loss of similar magnitude 12 . The excess diaphyseal apposition of bone tissue is reduced in ColXIII-overexpressing mice with the simultaneous lack of integrin α11 subunit, demonstrating the in vivo significance of ColXIII-induced signaling in bone turnover 8 . The role and function of ColXIII in the normal development of postnatal NMJs in Col13a1 −/− and Col13a1 tm/tm mice is well characterized 13,15,23,28 . The muscle tissue of Col13a1 −/− mice has reduced complexity and size of acetylcholine receptor (AChR) clusters already at the age of 4 weeks. In contrast to the Col13a1 −/− mice, the 12-week-old Col13a1 tm/tm mice have increased size and complexity of the AChR clusters compared to WT mice 23 .
Here, we show that the femoral bone area and cortical thickness are reduced in both the Col13a1 −/− and the Col13a1 tm/tm mice with aging, long after the occurrence of NMJ abnormalities. In contrast to opposing NMJ phenotypes of Col13a1 −/− and Col13a1 tm/tm mice, the reduction in femoral bone mass was similar in both mouse lines indicating that the reduction of bone mass is not secondary to the abnormal NMJ development, but rather due to a distinct role of ColXIII in bone tissue. We point out that according to marker gene expression bone 13,27 is in addition to NMJs one of the two tissues with highest ColXIII expression, and thus it is conceivable that the observed bone effects are direct causes of lack of this collagen. However, these data do not exclude the possibility of systemic effects caused by the lack of ColXIII. Additionally, we demonstrate here that the lack of soluble ColXIII in Col13a1 tm/tm mice causes a similar bone phenotype as the total knockout of Col13a1, suggesting the importance of soluble ColXIII in bone homeostasis. Previously, we have shown that ColXIII is mainly expressed by diaphyseal osteoblasts in the mouse femur 27 . This could be the cause of the predominant phenotypes in the diaphyseal area in all ColXIII mouse lines, as shown here for Col13a1 −/− , Col13a1 tm/tm , and previously for Col13a1 oe . While the alterations of the cortical metaphysis are not identical with those from the diaphysis, our results are consistent in the sense that cortical bone decreases with age in both anatomical locations. This is in line with prior research, which found that age-related cortical thinning differ between the metaphysis and diaphysis 29 . Previously we have shown that the soluble ColXIII, not the transmembrane form, activates JNK and ERK pathways in osteoblasts 12 . This finding is in accordance with lack of shedding having an impaired effect in the bone formation of the Col13a1 tm/tm mice as shown here. However, the changes in the Col13a1 −/− and the Col13a1 tm/tm mice are similar but not identical. The 72-week-old Col13a1 −/− mice have significantly lowered cortical and medullar areas but not a reduced cortical thickness whereas the Col13a1 tm/tm have a decreased cortical area and cortical thickness but an unaltered medullar area. Age-dependent cortical thinning in mice occurs when the loss of bone from the endosteal surface surpasses the quantity of bone deposited to the periosteal surface 29 . Previous studies demonstrate that this cortical thinning in mice is due to unbalanced endocortical a basic multicellular unit (BMU)-based remodeling, not osteoclastic modeling 29 . Based on our previous findings, soluble ColXIII affects specifically BMU-based remodeling 12 . The decrease observed here in the medullar expansion of the old Col13a1 −/− mice is consistent with these previous results, indicating the importance of ColXIII in BMU-based cortical remodeling. However, based on the data presented here, we cannot explain why in the Col13a1 tm/tm mice cortical thinning is increased without changes in the medullary expansion compared to WT. Nevertheless, the phenotypic similarity in bone of the Col13a1 −/− and Col13a1 tm/tm mice suggests that the soluble ColXIII is the main form affecting bone homeostasis, but also the transmembrane form appears to have some direct or indirect roles in bone remodeling.
Similarly to CMS19 patients 17,19 , the Col13a1 −/− and the Col13a1 tm/tm mice exhibit visible craniofacial malformations. However, the cephalometric analysis of the Col13a1 −/− and the Col13a1 tm/tm mice did not reveal a clear phenotypical pattern when both sexes were studied at two time points, though it revealed subtle and sexdependent alterations whose origin remain unclear. Moreover, some differences found in the 4 weeks old mice became nonsignificant at the 35-week time point, possibly because of delayed and abnormal postnatal development being overcome with age. Alterations of craniofacial morphology have been published in several mouse models of syndromic craniosynostoses. Whereas clear craniofacial abnormalities have been reported for example in mouse lines with mutations of FGFR1 30 , FGFR2 31-34 , TCF12 35 and EFNB1 36 , several mutant mouse models have been reported to present with only mild calvarial abnormalities compared to the respective human patients [37][38][39][40][41][42] . These mouse lines, however, often present with other skeletal abnormalities such as decreased femoral cortical thickness and cortical bone area [41][42][43] or decreased vertebral BV/TV and BMD 44 .
In conclusion, our findings in the skeletal tissues show that lack of ColXIII in mice indeed affects bone, although the findings are less pronounced than in the human disease, which is often the case also in other mouse models of human syndromes. Unlike the NMJ alterations, which stabilize with age in the Col13a1 −/− mice and the CMS19 patients 17,28 , the reduced bone phenotype becomes aggravated with aging of the mice. This finding may be significant also in understanding the effects of lack of ColXIII in humans. As such, our results further validate and expand the potential use of the Col13a1 −/− mice as a disease model for CMS19. Although the abnormal development of NMJs shows marked differences between the Col13a1 −/− and the Col13a1 tm/tm mice, www.nature.com/scientificreports/ we found the tubular bone mass to be similarly reduced in both mouse lines. This would indicate that especially soluble ColXIII has a significant role in the upkeep of cortical bone mass with aging, independent of its functions in normal NMJ development. As both complete and partial lack of ColXIII reduced femoral bone mass significantly, future research is warranted to assess the possibility of bone pathologies such as osteoporosis in human CMS19 patients.
Low-resolution micro-computed tomography (µCT). For µCT analyses, left femurs were dissected from male and female Col13a1 tm/tm and Col13a1 −/− mice at 4-, 12-, 25-, 35-and 72-week time points. Samples were also collected from age and gender matched WT littermate controls. Samples were stored in phosphatebuffered saline at -20 °C with no prior fixation. Most recent guidelines for µCT methodology were used 45 . Samples were imaged with a Skyscan 1176 scanner (Bruker microCT, Kontich, Belgium) designed for in vivo imaging of live animals. X-ray tube voltage of 50 kV with image pixel size of 8.71 μm and current of 500 µA with a 0.5 mm aluminum filter were used. One image projection was collected every 0.3° over 360° rotation with an exposure time of 4000 ms. Specimens were imaged without a scanning medium (i.e. air) wrapped in a moist paper and placed in a sealed container to prevent drying. Image slices were reconstructed into the tomographic image datasets using NRecon software (Skyscan, v. 1.6.5.2), where ring artefact and beam hardening corrections were applied. 2D and 3D volumetric analyses of datasets were done using CTAn software (Skyscan, v. 1.11.10.0). Both softwares were provided by the manufacturer. Regions of interest (ROIs) were manually drawn for trabecular and cortical bone quantitative morphometric analyses. Trabecular bone was assessed from distal femoral metaphyseal area using an irregular, anatomic region of interest drawn manually a few voxels away from the endocortical surface. Trabecular ROI continued 0.9 mm proximally starting from the distal growth plate. Following 3D morphometric parameters were measured: total volume of region of interest (TV), volume of the region segmented as bone (BV), bone volume fraction (BV/TV) and trabecular number (Tb.N). Diaphyseal cortical bone was assessed using a volume of interest starting 0.9 mm from femoral neck and extending distally towards the diaphysis. High-resolution µCT. For more detailed analysis of bone morphometric parameters, left mouse femurs were analyzed from Col13a1 −/− and Col13a1 tm/tm female mice using high-resolution computed tomography (HRCT). Samples from the 35-and 72-week time points were analyzed, and age-matched WT control samples were used. Skyscan 1272 (Bruker microCT, Kontich, Belgium) scanner, designed for high-resolution 3D imaging of ex vivo specimens, were used with an X-ray tube voltage of 50 kV, a current of 200 μA, an exposure time of 4000 ms and a 0.25-mm aluminum filter. X-ray images were acquired every 0.29° within a 360° rotation. Final image resolution was 1.75 μm. A sealed container and moist tissue paper were used to prevent sample drying during imaging. The image datasets were processed, and image artifact corrections were applied using NRecon software (v. 1.6.5.2). The datasets were reoriented using the Dataviewer software (v. 1.5.6.2) and morphometric analyses were conducted using the CTAn software (v. 1.11.10.0). All softwares were provided by the manufacturer. The reporting and discussion of the results followed current recommendations 45,46 . Trabecular bone was assessed from the distal femoral metaphyseal area using an anatomic ROI drawn a few voxels away from the bone endocortical surface. The length of the trabecular ROI was 0.7 mm (400 axial slices) starting from the distal growth plate and extending towards the diaphysis. Following 3D morphometric parameters were measured: TV, BV, BV/TV and Tb.N. Metaphyseal cortical bone was assessed using a 0.35-mm-long (200 axial slices) VOI starting 1.75 mm from the distal growth plate and extending proximally towards the diaphysis. Following 2D morphometric parameters were measured: Ct.Ar, Ma.Ar, and Ct.Th.
Cephalometric analysis. For cephalometric analyses, we imaged Col13a1 −/− and Col13a1 tm/tm mouse skulls at 4-and 35-week time points using HRCT. Both genders were studied, and age-and gender-matched WT littermate controls were used. The samples were stored at − 20 °C in phosphate-buffered saline (PBS) without prior fixation. The samples were imaged using a Skyscan 1272 (Bruker microCT, Kontich, Belgium) scanner with a tube voltage of 50 kV and 0.5 mm aluminum filter. X-ray images were collected every 0.29° within a 360° rotation with an exposure time of 1550 ms. The final image resolution was 10.7 μm. The samples were covered in moist tissue paper in a sealed container during imaging to prevent drying. Image processing was done using the NRecon software (v. 1.6.5.2) and the datasets were reoriented using the DataViewer software (v. 1.5.6.2). Softwares were provided by the manufacturer. An in-house MATLAB (R2018b, The MathWorks, USA) script was used to perform the cephalometric analyses (Supplementary material and Supplementary Figs. S4-S6). A sagittal and transverse projection image of 3D reconstructed samples was produced from each dataset and saved with a corresponding filename. Measurements for craniofacial, maxillary, and mandibular morphology were con- www.nature.com/scientificreports/